Our objectives are to define the mechanism of action and regulation of the enzyme phosphoenolpyruvate (PEP) carboxylase of Escherichia coli, and the structural requirements for those functions. It is becoming apparent that enzymes may assay normally when studied in vitro but function abnormally in vivo due to mutations that have altered their regulatory properties. PEP carboxylase is an enzyme which may behave in this manner and is being used as a model for these studies. Indeed, an E. coli mutant deficient in regulation of PEP carboxylase has been described. This mutant has altered metabolism and it grows very slowly. These studies are designed to characterize as well as possible, all functions of this enzyme and the structures involved in those functions. This phase of the project is designed to: (1) Obtain information on the mechanism by which fru-1,6-P2 leads to hysteresis in the expression of catalytic activity and increases cooperativity between subunits. (1) Try to obtain some direct evidence for the existence of a carboxylphosphate intermediate in the catalytic mechanism of this reaction. (3) Determine the potential importance of the interactions of guanosine-5'-diphosphate-3'-disphosphate and guanosine-5'-diphosphate-3'-monophosphate with phosphoenolpyruvate carboxylase by determining the kinetics of their interactions with the wild-type and mutant enzymes, and (4) To continue structure-function analyses by characterization of additional mutants and determining structural feature as necessary. Kinetics, NMR, gene cloning, and protein modification and structural analyses are among the techniques to be used.